Diagnosis and Treatment of Cancer Expressing ILT3 or ILT3 Ligand

ABSTRACT

The present invention relates to methods of using the expression of ILTL3 ligand or ILT3 on certain types of cancer cells as a diagnostic tool. Methods are provided for treating ILT3-ligand expressing cancers, such as T-cell acute lymphoblastic leukemia (T-cell acute lymphoblastic leukemia), for example by administering ILT3, the extracellular domain of ILT3 or ILT3Fc conjugated to a cytotoxic agent to kill the targeted cancer cell. Other methods are provided for treating cancers that express ILT3 on their surface, such as monocytic forms of AML, for example by administering anti-ILT3 antibodies conjugated to a cytotoxic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 14/342,440, filed Nov. 7,2014 which is a 371 national stage application that claims benefit ofPCT/US12/53714, filed Sep. 4, 2012, entitled, “Diagnosis and Treatmentof Cancer Expressing ILT3 or ILT3 Ligand,” and claims benefit of thefollowing U.S. Provisional Applications 61/590,510, filed Jan. 25, 2012,entitled, “Diagnosis of Acute Lymphoblastic Leukemia” and, 61/530,936,filed Sep. 2, 2011, entitled, “Diagnosis and Treatment of HumanMalignancies Using mAb Anti-ILT3 or ILT3Fc”, the entire contents ofwhich are hereby incorporated by reference as if fully set forth herein,under 35 U.S.C. §119 (e).

BACKGROUND OF THE INVENTION

Acute lymphoblastic leukemia (ALL) is a form of leukemia that ischaracterized by the presence of excess lymphoblasts due to thecontinuous multiplication of malignant immature white blood cells. ALLis a disease that rapidly progresses and is characterized by crowding ofnormal cells in the bone marrow due to the continuous multiplication ofimmature white blood cells.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) causes 15% ofacute leukemias in childhood, and approximately 40% of lymphomas inchildhood. Acute refers to the relatively short time course of thedisease, as it can be fatal in as little as a few weeks if leftuntreated. Most common in adolescent males, T-ALL's morphology isidentical to that of precursor B-cell lymphoblastic leukemia. The rapidprogression or relatively short time course of T-ALL without treatmentmakes early diagnosis extremely important since early intervention maydelay onset of the disease and ultimately increase survival rates.

Currently no cellular markers specific for acute T-ALL are known, makingdiagnosis of the disease difficult. The only effective diagnostic methodfor acute T-ALL includes medical history, physical examination, completeblood count, and blood smears. There is an unmet need for theidentification of cellular markers specific for acute T-ALL. Thedevelopment of a diagnostic cellular marker that could aid in thediagnosis and treatment of T-ALL in its early stages is desirable.

Acute myeloid leukemia (AML) is one of the most common types of leukemiain adults (50), (51). Despite major advances in our understanding of thebiology of AML, the 5-year survival of AML patients is only 20-40%. Ithas been proposed that AML originates from self-renewing hematopoieticstem cells (HSC)/progenitors that have acquired multiple genetic and/orepigenetic changes (52). These cells initiate a developmental hierarchyof single- or multiple lineage precursors exhibiting various degrees ofmaturation arrest. The heterogeneity of AML is evident from the widevariety of clinical manifestations, response to therapy, phenotypicfeatures, and molecular and cytogenetic alterations (53), (54), (55). Inclinical practice, the accurate diagnosis of AML subtypes is essentialfor risk stratification and treatment planning. The World HealthOrganization (WHO) and French American British (FAB) classificationsystems are currently used to subtype AML (50), (51), (56). Although theWHO classification system relies heavily on cytogenetic findings, thesedata are often not available at the time of diagnosis and initiation oftreatment. Furthermore, the most frequent cytogenetic feature, which isidentified in over 40% of the patients with AML, is the lack of anychromosomal alterations (57), (58). In patients with cytogeneticallynormal AML, specific gene mutations have been associated with certainAML subtypes (59), (60).

Therefore there is a need not only for better markers to distinguish thevarious forms of AML, but more specific treatments for the various formsof AML.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that certain forms ofcancer express ILT3 on their surface, including chronic lymphocyticleukemia (CLL) and AML with monocytic differentiation (AML m4/m5). Otherforms of cancer have been discovered to express ILT3 ligand on theirsurface, such as T-ALL. Certain embodiments relate to methods andcompositions for diagnosing and treating these forms of cancer.

In certain embodiments, cancers expressing ILT3 like CLL and AMLm4/m5with monocytic differentiation are diagnosed and distinguished fromnoncancerous cells or other forms of cancer by binding to an anti-ILT3antibody that is detectably labeled, or that can be detected by bindingto a secondary reagent that is detectably labeled. Detectable labels foruse in embodiments of the invention are described below. In anembodiment, diagnosing ILT3-expressing cancers includes (a) obtaining abiological sample from a subject that has or may have cancer thatexpresses ILT3 on the cancer cell surface, and a control sample from anormal subject; (b) contacting the subject and control samples with ananti-ILT3 antibody or a portion thereof that selectively binds to ILT3under conditions that permit the probe to bind to the ILT3 expressed onthe cancer cell surface; (c) determining the number or percentage ofcancer cells that are bound to the antibody in the subject and controlsamples; and (d) determining that the subject has the ILT3-expressingcancer, if the number or percentage of cells bound to the antibody inthe subject sample is significantly higher than the corresponding numberor percentage of cells bound to the antibody in the control sample.

Certain embodiments are directed to treating these ILT3-expressingcancers by administering therapeutically effective amounts of anantibody that selectively binds to the ILT3 on the surface of the cancercells (including newly discovered anti-ILT3 mAbs A, B, C and D describedherein), which antibody is linked either to an isotope that emitsradiation at a level that kills the cancer cell, or to a cytotoxicagent, thereby treating the subject for the cancer. An exemplaryembodiment is a) identifying a subject in need of treatment for a cancerthat expresses ILT3 on its surface, and b) administering therapeuticallyeffective amounts of an antibody that selectively binds to the ILT3 onthe surface of the cancer cells, which antibody is linked either to anisotope that emits radiation at a level that kills the cancer cell, orto a cytotoxic agent, thereby treating the subject for the cancer.

Other embodiments are directed to methods for diagnosing anddistinguishing cancers that express ILT3 ligand on their surface such asT-ALL, from noncancerous cells or other forms of cancer by (a) obtaininga biological sample from a subject that has or may have cancer thatexpresses ILT3 ligand on the cancer cell surface, and a control samplefrom a normal subject; (b) contacting the subject and control sampleswith a probe that selectively binds to ILT3 ligand under conditions thatpermit the probe to bind to the ILT3 ligand on the cancer cell surface;(c) determining the number or percentage of cancer cells that are boundto the probe in the subject and control samples; and (d) determiningthat the subject has the ILT3 ligand-expressing cancer, if the number orpercentage of cells bound to the probe in the subject sample issignificantly higher than the corresponding number or percentage boundin the control sample. In certain embodiments the probe that selectivelybinds to ILT3 ligand is ILT3, ILT3Fc, the extracellular domain of ILT3or an ILT3 ligand-binding fragment of one of these molecules.

In other embodiments cancers expressing ILT3 ligand on their surface,like T-ALL are treated by a) identifying a subject in need of treatmentfor a cancer that expresses ILT3 ligand on its surface, and b)administering therapeutically effective amounts of ILT3, ILT3Fc, theextracellular domain of ILT3 or a fragment thereof comprising the ILT3ligand binding site, which ILT3, ILT3Fc, extracellular domain andfragment is linked either to a radioactive isotope that emits radiationat a level that kills the cancer cell or to a cytotoxic agent, therebytreating the subject for the ILT3 ligand-expressing cancer.

Cytotoxic agents for use in embodiments of the invention include Taxol®,Pseudomonas exotoxic fragment, cytocalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etopside, tenopside, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy antracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosteron,glycocorticoids, procain, tetracaine, lidokaine, propranolol, andpuromycin. Alternatively, the cytotoxic agent is the CD3 chain of the Tcell receptor complex and whereby cancer cell lysis is induced byT-cell-mediated cytotoxicity against the cancer cell.

Other embodiments are directed to a diagnostic kit for the detection ofcancer cells that express ILT3 on their surface, comprising an isolatedmonoclonal antibody or an ILT3-binding portion thereof according toclaim 1; in another embodiment, the monoclonal antibody or portionthereof is detectably labeled either with a radioisotope or a label thatcan be visualized. Another embodiment includes a diagnostic kit for thedetection of cancer cells that express ILT3 ligand on their surface,comprising ILT3, ILT3Fc, extracellular domain of ILT3, or an ILT3ligand-binding fragment thereof. In an embodiment the ILT3, ILT3Fc,extracellular domain of ILT3, or the ILT3 ligand-binding fragment isdetectably labeled either with a radioisotope or a label that can bevisualized. Other embodiments are directed to newly discovered isolatedanti-ILT3Fc monoclonal antibodies designated A, B, C or D or anILT3-binding portion thereof, especially for use in embodiments of theinvention to identify and treat cancer expressing ILT3.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1A and FIG. 1B are graphs demonstrating bone marrow aspirate from apatient with T-ALL (CD45 dim gate identifies leukemic cells).

FIG. 2A and FIG. 2B are graphs illustrating a T-ALL cell line expandedand maintained in culture.

FIG. 3A and FIG. 3B are graphs showing normal (resting) T cells fromhealthy individuals that express low or no ILT3 ligand. Specifically,CD4-gated T helper cells from a healthy blood donor indicate 1% ILT3Fcbinding.

FIG. 4A and FIG. 4B are graphs demonstrating CD3-gated T cells from ahealthy blood donor with 12% ILT3Fc binding.

FIG. 5A and FIG. 5B. Flow cytometric analysis of whole bone marrowaspirate obtained from a patient with no evidence of hematologicaldisease. FIG. 5A shows all data. FIG. 5B shows graphs of the dataseparated by CD45 brightness (increasing from dim on left to bright onright, with the remainder, called ungated, in the middle). The data arealso separated by side scattering (SSC) levels, with higher levels inthe graphs at the top and lower levels in graphs at the bottom. Cellswere gated in the CD45 versus side scatter plot as follows:Lymphocytes—CD45bright/low SSC, Monocytes—CD45bright/intermediate SSC,Granulocytes—CD45dim/high SSC, Precursor cells—CD45dim/low tointermediate SSC. Cell surface expression of ILT3 and CD 14, or CD33,CD34 or CD 117 is depicted. The results are representative for theimmunophenotypic profile observed in 20 non-involved bone marrowsamples.

FIG. 6 is a bar graph that illustrates expression of relevant cellmarkers analyzed by flow cytometry in 37 patients with acute myeloidleukemia.

FIG. 7A, FIG. 7B, and FIG. 7C. A flow cytometric analysis of whole bonemarrow aspirates obtained from patients with AML. Leukemic cells weregated based on abnormal immunophenotypic and light scatter features. Theresults are representative for: AML m4/m5 (M4/M5—Panels A-D), AMLwithout differentiation (M 11M2—Panel E), and APL (M3—Panel F). Cellsurface expressions of ILT3, CD14, CD34, CD45 and CD117 are depicted.FIG. 7A are graphs that show results for CD45 vs SSC in the variouspanels; FIG. 7 B are graphs that show results for CD14 vs ILT3 in thevarious panels; and FIG. 7C are graphs that show results for CD34 vsCD117 in the various panels.

FIG. 8 is a graph that illustrates Kaplan-Meier curves illustratingpatient survival within ILT3+ and ILT3− AML groups.

In the Summary of the Invention above, in the Detailed Description ofthe Invention, and the claims below, as well as the accompanyingfigures, reference is made to particular features of the invention. Itis to be understood that the disclosure of the invention in thisspecification includes all possible combinations of such particularfeatures. For example, where a particular feature is disclosed in thecontext of a particular embodiment or embodiment of the invention, or aparticular claim, that feature can also be used, to the extent possible,in combination with and/or in the context of other particularembodiments and embodiments of the invention, and in the inventiongenerally. For the purposes of explanation, numerous specific detailsare set forth in order to provide a thorough understanding of thepresent invention. It will be apparent, however, to one skilled in theart that the present invention may be practiced without these specificdetails.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry, and immunology,which are within the skill of the art. Unless defined otherwise,technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

It has been discovered that cancer cells of AML type with monocyticdifferentiation (AML m4/m5) expresses ILT3 on their surface. Cancercells of another form of cancer, the T-ALL form, have been discovered toexpress ILT3 ligand on their surface. Certain embodiments relate tomethods and compositions for diagnosing and treating these differentforms of cancer; those that express ILT3 and those that express ILT3ligand.

I. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics, protein, and nucleic acid chemistry and hybridizationdescribed herein are those well-known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed through the present specification unless otherwise indicated.Many references are available to provide guidance in applying the abovetechniques (Kohler et al., Hybridoma Techniques (Cold Spring HarborLaboratory, New York, 1980); Tijssen, Practice and Theory of EnzymeImmunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal AntibodyTechnology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal HybridomaAntibodies: Techniques and Applications (CRC Press, Boca Raton, Fla.,1982); and Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987)). Northern blot analysis is aconventional technique well known in the art and is described, forexample, in Molecular Cloning, a Laboratory Manual, second edition,1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 SkylineDrive, Plainview, N.Y. 11803-2500. Typical protocols for evaluating thestatus of genes and gene products are found, for example in Ausubel etal. eds., 1995, Current Protocols In Molecular Biology, Units 2(Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18(PCR Analysis).

“AML with monocytic differentiation” hereafter “AMLm4/m5” means subtypesof acute myeloid leukemia that comprise over 10% of AML cases and occursin all age groups. They have been found frequently associated withdeletions and translocations involving 11q23, inv(16) and t(9,11), andmay also harbor t(6,9), inv(3), NPM1 or FLT3-ITD mutations. Monocyticdifferentiation is identified by morphology and confirmed bycytochemical stains or flow cytometry.

“Antibody-linked cytotoxic agent” means a monoclonal antibody (mAb) thatis linked, conjugated, or otherwise bound to a cell-killing drug to beused as vehicle to target cancer cells due to the high bindingspecificity.

“Biological sample” means a variety of sample types obtained from anorganism and can be used in the embodiments of the herein describeddiagnostic or monitoring assays. The sample is selected from any part ofa patient's body, including, but not limited to, blood, lymph nodes,spleen, or bone marrow aspirates. Preferred samples for diagnosingcancers that express ILT3 or ILT3 ligand are blood (including plasma andserum), bone marrow aspirates, fine needle aspirates, body fluids (suchas pleural fluid, cerebro-spinal fluid). The term encompasses samplesthat have been manipulated in any way after their procurement, such asby treatment with reagents, solubilization, or enrichment for certaincomponents. The term encompasses a clinical sample.

“ILT3” means “Immunoglobulin-Like Transcript-3”, and is synonymous with“ILT-3”, “LIR-5”, “CD85K” and “LILRB4.” The mRNA coding sequence forhuman ILT3 is provided under GenBank No. U82979. Human ILT3 is atransmembrane protein having 447 amino acids with a predicted molecularmass of about 47 kD. ILT3 behaves as an inhibitory receptor whencross-linked to a stimulatory receptor. ILT3 has an extracellular regionthat includes N-terminal amino acids 1-259 and a signal peptide of aminoacids 1-16; a transmembrane domain that includes amino acids 260-280;and a cytoplasmic domain that includes amino acids 281-448. ILT3 hascytoplasmic domain which includes an ITIM motif at amino acids 412-415and 442-445. The extracellular domain of contains two Ig domains. “ILT3”shall mean the gene, mRNA, or protein of “Immunoglobulin-LikeTranscript-3”, and is synonymous with “ILT-3”, “LIR-5”, “CD85K” and“LILRB4”. The mRNA coding sequence for human ILT3 is provided underGenBank No. U82979.

The “extracellular domain of ILT3” or “ED” means the N-terminal 258amino acid residues of ILT3 (e.g., human ILT3 having the sequence ofGenBank Accession No. U82979). The extracellular domain of contains twoIg domains, one or both of which are likely to contribute to the ILT3ligand binding. The extracellular domain of ILT3 includes, for example,the IgG1-like domain 1 (residues 42-102 of human ILT3), the IgG1-likedomain 2 (residues 137-197 of human ILT3), and the N-terminal 250, 240,230, 220, 210, 200, 190, 180, 170, 160 or 150 amino acid residues ofILT3.

“ILT3 Ligand” means the molecule expressed on the surface of T-cells andcertain cancer cells such as T-ALL cells to which ILT3, ILT3-FC andcertain fragments thereof selectively bind. The ILT3 ligand is expressedtransiently on the surface of up to about 10-30% of normal T cells fromperipheral blood monocytes (PBMC) which have been allo-activated byexposure to HLA mismatched cells. The ligand-binding site on ILT3 is inthe extracellular domain of ILT3.

“Specific binding of antibodies” means that the antibodies: 1) exhibit athreshold level of binding activity, and/or 2) they do not significantlycross-react with known related polypeptide molecules.

“Specific binding” of an agent, such as the ILT3 ligand-binding probemeans that the agent binds to the target protein, such as ILT3 ligand,with greater affinity than it binds to unrelated antigens.

“ILT3Fc” means the extracellular domain of human ILT3 (ED) operablyaffixed to the Fc portion of an immunoglobulin. In an embodiment the Fcportion comprises a function-enhancing mutation, such as a mutation thatinhibits the binding of the Fc portion of an immunoglobulin to an Fcreceptor. In an embodiment the Fc portion derived from human IgG1. Inone example, the function-enhancing mutation in the Fc portion of theimmunoglobulin is an Asn-->Gln point mutation at amino acid residue 77of the Fc portion of human IgG1. The Fc portion of ILT3Fc may besubstituted with any other peptide that promotes dimerization oroligomerization of the probe or otherwise stabilizes the probe. Forexample, the peptide may comprise cysteine residues that form disulfidebonds or other residues that promote covalent or nonconvalentinteractions between the peptides such that the peptides mediatedimerization or oligomerization. Exemplary oligomerization domains aredescribed in, e.g., WO 00/69907, WO 99/62953, WO 98/56906, WO 98/18943,and WO 96/37621.

“ILT3 probe” and “ILT3 ligand-binding probe” are used interchangeable tomean a molecule that selectively binds to ILT3 ligand. The ILT3ligand-binding probes include full-length ILT3, the extracellular domain(ED) of ILT3, the recombinant protein (ILT3Fc), and fragments of ILT3that include the ED. The probes can be used alone or they can be boundto a compound that stabilizes the probe or increases binding of theprobe to the targeted ILT3 ligand. Since there is a high level ofsequence homology among various species, the ILT3 ligand-binding probes,though preferably including or derived from human ILT3, can come fromany species as long as it selectively binds to ILT3 ligand on a targetedcancer cell or T-cell.

“A probe for detecting ILT3” means a molecule that specifically binds toILT3 that is expressed on the surface of a cancer cell, such as an AMLcancer cell of monocytic lineage. Such probes include anti-ILT3 mono- orpolyclonal antibodies, or an antigen (ILT3)-binding portion thereof.

“Detectable probe” means a probe for use in the diagnostic methodsdescribed herein, that may be detected or visualized using well knownmethods such as radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H,enzymes, chemiluminescent agents, and fluorescent dyes. Different typesof chemical labels or tags can be conjugated to secondary or primaryantibodies against ILT3 or ILT3Fc to facilitate their visualization(i.e., detection and measurement.) The choice of label or tag depends onthe sensitivity required, ease of conjugation with the probe, stabilityrequirements, and available instrumentation.

“Immunoglobulin” and “antibody” are used synonymously herein, andinclude any anti-ILT3 antibody that has high affinity for ILT3,including those antibodies that have high affinity for the extracellulardomain of ILT3. In the context of the invention, anti-ILT3 antibodiesare administered therapeutically to target delivery of a cytotoxin to acancer cell expressing ILT3 on its surface (such as AML m4/m5). Fordiagnostic use, the anti-ILT3 antibodies selectively bind to ILT3expressed on the surface of a cancer cell such as AML m4/m5. Included,by way of example, are both naturally occurring and non-naturallyoccurring antibodies., polyclonal and monoclonal antibodies, anyantigen-binding fragments (e.g., Fab fragments, as opposed to Fcfragments) thereof, chimeric antibodies (e.g., humanized antibodies) andwholly synthetic antibodies, and antigen-binding fragments thereof.Within the scope of the term “antibody” are antibodies that have beenmodified in sequence, but remain capable of specifically binding toILT3, ILT3Fc or the ED or fragment of ILT3 comprising the ED. In someembodiments anti-ILT3 antibodies are used as secondary reagents to labelILT3 (used as a probe) that binds to ILT3 ligand. Examples of modifiedantibodies include interspecies chimeric and humanized antibodies;antibody fusions; and heteromeric antibody complexes, such as diabodies(bispecific antibodies), single-chain diabodies, and intrabodies (see,e.g., Marasco (ed.), Intracellular Antibodies: Research and DiseaseApplications, Springer-Verlag New York, Inc. (1998) (ISBN: 3540641513),the disclosure of which is incorporated herein by reference in itsentirety).

By “French American-British (FAB) subtypes” is meant the FAB subtypesM4, M5a and M5b″ of AML that are morphologically characterized bymonocytic differentiation. These subtypes show distinct clinicalfeatures, such as high risk of extramedullary disease, high leukocytecount, and coagulation abnormalities. AML with monocytic differentiationcomprises (herein AML m4/m5) over 10% of AML cases and it occurs in allage groups.

The terms “individual,” “subject” and “patient,” are usedinterchangeably herein and refer to any human subject for whomdiagnosis, treatment, or therapy is desired.

“Operably affixed” or “bound to” or “linked to” or “conjugated to” withrespect to the connection between a probe (an ILT3 ligand-binding probethat includes ILT3, ILT3Fc and the ED, or a probe for detecting ILT3such as an anti-IL3 antibody) and the label, or the probe and Fc, forexample, shall mean affixed (e.g., via peptide bond) in a mannerpermitting the ILT3 ligand-binding probe to bind to the ILT3 ligand onthe surface of T-cells or for an anti-ILT3 antibody to bind to ILT3. Inone embodiment, a polypeptide linker of 10, 11, 12, 13, 14, 15 or 16amino acid residues in length is used to join the ILT3 and Fc moieties.

“Polypeptide” and “protein” are used interchangeably herein, and eachmeans a polymer of amino acid residues. The amino acid residues can benaturally occurring or chemical analogues thereof. Polypeptides andproteins can also include modifications such as glycosylation, lipidattachment, sulfation, hydroxylation, and ADP-ribosylation.

“Selectively binds” and “specifically binds” are used interchangeably tomean the specific or preferential affinity with which two or moreproteins interact such as an antibody or a protein with a substrate. Alabeled ILT3 ligand-binding probe may specifically bind to the T-ALLbiomarker ILT3 ligand.

“Significantly higher” with respect to the level of detectably labeledT-cells or cancer cells means about one standard deviation above themean of a normal population, which may vary depending on the sample sizeof the normal and cancer populations and the type of cancer.

In the following description, for the purposes of explanation, numerousspecific details are set forth in order to provide a thoroughunderstanding of the present invention. It will become apparent,however, to one skilled in the art that the present invention may bepracticed without these specific details.

II. OVERVIEW

The inhibitory receptor ILT3 is a member of the immunoglobulin-liketranscript (ILT, LIR or LILR) family and is expressed by dendriticcells, monocytes, endothelial cells and osteoclasts, but not Tlymphocytes (47), (48), (49). Encoded in the leukocyte receptor clusteron human chromosome 19, the ILT proteins are structurally andfunctionally related to the killer cell immunoglobulin-like receptors(KIR) and deliver either activating or inhibitory signals (50), (51),and (52). Inhibitory ILT proteins display intracytoplasmic ITIM motifsthat recruit SHP-1 phosphatases that contribute to downstream inhibitorysignals. In contrast, activating ILTs have short cytoplasmic tails andassociate with adaptor proteins, such as FceRIg, to activate cellsignaling.

The function of ILT3 proteins expressed by antigen-presenting cells(APC), such as monocytes and dendritic cells, has been described (53),(54), (55), (56). For example, dendritic cells expressing high levels ofinhibitory receptors ILT3 and ILT4 were shown to induce anergy of CD4+Thelper cells and differentiation of CD8+T suppressor cells (53), (54),and (51). On the other hand, knockdown of ILT3 renders dendritic cellsmore sensitive to Toll like receptor (54). Although absent on normal Blymphocytes, ILT3 is expressed by leukemic B cells from a subset of CLLpatients displaying extensive lymph node involvement. Expression of ILT3by hematopoietic precursors has not been yet characterized. It has nowbeen discovered that ILT3 is selectively expressed on AML of monocyticdifferentiation.

U.S. application Ser. No. 12/072,119, entitled ILT3 and ILT4-RelatedCompositions and Methods, discloses a means for treating cancer byremoving sILT3 from the blood either and then returning cleansed bloodto the patient in order to increase the immune response to a diseasesuch as a viral disease or cancer. This method can further includeadministering an anti-sILT3AB to neutralize sILT3. However, there is norecognition of that there is any form of cancer that expresses ILT3 onits surface, nor is there a disclosure or suggestion that an anti-ILT3antibody could be conjugated to a toxic agent to kill a cell, such as acancer cell that expresses ILT3. Moreover, while ILT3 on the surface ofa cancer cell could bind the anti-ILT3 mAb, this alone is not enough tokill a cancer cell. (Chang et al. Nat Immunol 2002).

In PCT/US02/20128 entitled ILT3 and ILT4-Related Compositions andMethods, a method for treating cancer by administering an anti-ILT3antibody is disclosed. It explains that “the inhibitory function of ILT3and ILT4 receptors concerns the central control mechanisms of the immuneresponse which must be inhibited to induce specific tolerance intransplantation and autoimmune diseases and augmented [the immuneresponse] in AIDS and Cancer.” Nowhere does it suggest that an anti-ILT3antibody can bind to a cancer cell itself, nor does it suggestadministering an anti-ILT3 antibody conjugated to a cytotoxic agent tokill the targeted ILT3-expressing cancer. Instead, this applicationdiscloses methods that enhance the immune response by blocking immunesuppression by neutralizing ILT3 thereby permitting the cancer patientto mount a stronger immune response to the cancer.

ILT3-Ligand Expressing Cancers

It is known that the ILT3 ligand (herein “ILT3 ligand” or “ILT3L” or“the ligand”) is expressed on the surface of certain cells. However, thesequence or chemical identity of the ILT3L has not yet been identifiedand so it is not yet possible to generate antibodies that selectivelybind to the ligand. The ILT3 ligand by definition, selectively binds tothe inhibitory receptor ILT3. It is also known that the ligand-bindingsite on ILT3 is in the extracellular domain of ILT3. Further, ILT3ligand is expressed transiently on the surface of up to about 15% ofnormal T cells.

It has now been discovered that the ILT3 ligand is constitutively andselectively expressed at high levels on at least 50% and typically 90%of malignant T-ALL cells harvested from patients known to have thedisease, as well as in T-ALL cell lines. As is discussed below, thisdiscovery is the basis of new methods for diagnosing and treating T-ALLand any other cancer that is discovered to express ILT3-ligand on itssurface.

ILT3-Expressing Cancers

Immunohistochemical analysis is often used to subtype AML yet its valuein the diagnosis of AML with monocytic components is limited by the lackof highly sensitive and specific monocytic markers (40). (41). Severalmarkers are being used for ascertaining monocytic differentiation byflow cytometry, e.g. CD4, CD11c, CD14, CD36 and CD64 (44), (45), (46).Although the expression of these markers by the leukemic cells ishelpful for lineage assignment, the diagnosis of AML m4/m5 remainschallenging.

The French American-British (FAB) subtypes M4, M5a and M5b thatdistinguish AMLm4/m5 with monocytic differentiation, show distinctclinical features, such as high risk of extramedullary disease, highleukocyte count, and coagulation abnormalities (36), (37). AMLm4/m5comprises over 10% of AML cases and it occurs in all age groups (25),(36), and (39). Certain trans locations, such as t(9, 11) and 16q22,involving the mixed lineage leukemia (MLL) and the core binding factorbeta genes, respectively, are commonly seen in monocytic leukemias (26),(33). Mutations of the nucleophosmin (NPM1) gene have been alsoassociated with myelomonocytic or monocytic morphology and arepredictive of favorable outcome (30). However, these abnormalities arenot absolutely specific for the monocytic lineage and identify only afraction of patients with AML displaying monocytic differentiation.

It has now been discovered that ILT3 is expressed by normal and leukemicmyeloid precursor cells, and that ILT3 expression is therefore abiomarker for identifying normal hematopoietic precursors committed tothe monocytic lineage and for distinguishing AML m4/m5 from other typesof AML. It has also been previously shown that ILT3 is a useful markerfor identifying (CLL) (57). This discovery is the basis for new methodsfor diagnosing and treating AML and CLL cancers that express ILT3, andany other cancer that is discovered to express ILT3 on its surface.

III. RESULTS ILT3 Ligand is Constitutively and Selectively Expressed atHigh Levels on Malignant T-ALL Cells

12/12 T-ALL patients exhibited ILT3 ligand expression on malignantleukemic cells in a blood sample. Malignant leukemic cells wereclinically diagnosed as such using flow cytometry, cytopathology andkaryotyping and identified by in flow cytometry experiments by theCD45-dim/negative expression and low SSC profile. In one of the 12patients, the ligand was expressed on 50% of the malignant T-ALL cells,but in the other 11 patients the ligand was expressed on 90% of theT-ALL cells. By contrast, only a small fraction of normal T-cellsexpressed the ligand (about 15%). Based on this discovery, the ILT3ligand is a clinically useful biomarker for diagnosing patients who haveT-ALL. The expression of ILT3 ligand also differentiated T-ALL fromother forms of leukemias.

In view of this discovery, a set of embodiments is directed to a methodfor diagnosing ILT3 ligand-expressing cancers including T-ALL in apatient by determining if a biological sample taken from a patientincludes malignant T-ALL cells that express ILT3L at significantlyhigher levels that T-cells from a normal control patient. Bysignificantly higher is meant that the number or percentage of ILT3ligand-expressing T cells in a patient is about one standard deviationor more above the mean seen in a normal patient or population of normalpatients.

In an embodiment, this diagnosis of T-ALL is determined by obtaining abiological sample from a patient suspected of having T-ALL and anothersample from a normal subject; contacting the T-cells in a test samplefrom the patient and in a control sample from the normal subject, withdetectably labeled ILT3 ligand-binding probe (such as ILT3, the ED ofILT3, or preferably fluorescently labeled ILT3Fc); determining thenumber [or percentage] of ILT3 ligand-positive T-cells in each sample,and determining that the patient has T-ALL if the number [or percentage]of labeled T-cells labeled in the test sample is significantly higher(at least about 1 standard deviation above normal) than thecorresponding number or percentage detected in the control sample. Inthe example samples from 12 patients used for this study significantlyhigher labeling was about 15% higher in T-ALL patients than in normalpatients. This actual percentage used in a particular embodiment maychange based on sample size of the normal and cancer populations and onthe type of cell expressing ILT3.

In an embodiment, the detectable label on the ILT3, ED, or ILT3Fc probeis a fluorophore and the amount of label bound to the T-cells in thetest and control samples of step (d) above is determined using flowcytometry. The method may further comprise quantifying the amount offluorescence emitted from each fluorophore-labeled T-cell, for example,using flow cytometry.

In some embodiments of the method, the ILT3 ligand-binding probe (ILT3,ILT3Fc, or ED or fragment thereof) is indirectly labeled with asecondary reagent such an anti-ILT3 antibody (monoclonal, polyclonal, orchimeric antibody) that is detectably labeled. Further details of theassay where the probe is indirectly labeled are illustrated bynon-limiting Example 1. Four new anti-ILT3 monoclonal antibodies havebeen discovered and are herein referred to as anti-ILT3 mAb A, B C andD. Certain embodiments of the invention are directed to these newanti-ILT3 mAbs and their diagnostic use for example to detectILT3-ligand expressing cancer cells. In such an embodiment the ILT3ligand complexes with ILT3, ILT3Fc or ED probes, and the anti-ILT3antibodies bind to the probe. In some embodiments discussed below, theanti-ILT3 mAbs are conjugated to cytolytic/cytotoxic agents in order tokill ILT3-expressing cancer cells such as AML and CLL. In otherembodiments the anti-ILT3 mAb are detectably labeled and used todiagnose cancers that express ILT3 on their surface, such as T-ALL andCLL.

Typical labels for use in detecting proteins and antibodies includelabeling a protein or antibody for detection or visualization usingradioactive isotopes, enzyme substrates, metal complexes, haptens,co-factors, ligands, colorimetric agents, dyes, and chemiluminescent orfluorescent agents that can be bound to the protein or antibody. Thefluorescent agents are selected from the group comprising 6-FAM™,Acridine, Alexa dye, e.g., Alexa 488, Alexa Fluor, AMCA, BODIPY, CascadeBlue, Cholera toxin B, Cy2, Cy3, Cy5, Cy5.5, Cy7, Dabcyl, Dansylderviatives, Diamidino yellow, Dy 750, Edans, Eosin, Erythrosin, Fastblue, Fluorescein isothiocyanate, Green fluorescent protein (GFP) andderivatives, HEX™, Horseradish peroxidase, Hydroxystilbamidine, IowaBlack®, IRDye®' Joe, LightCycler 640, MAX 550, Oregon Green,Phycoerythrin, Pseudorabies virus, Red Leuco dye,3,3′,5,5′-tetramethylbenzidine (TMB), Rhodamine and its derivatives,e.g., Rhodamine Green™ and Rhodamine Red™, Rhodol Green, ROX™, TET™, TEX615, Texas Red °, TYE (including TYE™ 563, TYE™ 665, TYE™ 705),Umbelliferone, WellRED™ D2, WellRED™ D3, WellRED™ D4 and TAMRA dyes(such as pyrene, TAMRA, FITC), or combinations thereof. GFP was used inthe experiments described herein, however there are now many differentmutants of GFP (e.g., see Shaner N, Steinbach P, Tsien R “A guide tochoosing fluorescent proteins” (PDF). Nat Methods v2 (12), pp 905-9,2005). Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, for example in Sambrooket al., Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989;and Ausubel et al. Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998.

Direct labeling methods include radioisotope labeling. Radiolabelsinclude, but are not limited to ¹³¹I, ¹⁴C, ⁴⁵Ca, and ³H, ¹²⁵I, ³²P and³⁵S. Indirect labeling methods include binding fluorescent tags, orbiotin complexes (which can be bound to avidin or streptavidin), orpeptide or protein tags. Visual detection methods include colorimetricagents, dyes, chemiluminescent or fluorescent agents, or with horseradish peroxidase, alkaline phosphatase and the like. Any label that canbe detected can be used to quantify binding of Fluor the ILT3ligand-binding probe (comprising ILT3, or ILT3Fc or ED) to ILT3 ligandon T-cells. Fluorophores are typically operably affixed, bound orotherwise attached to the probe or the secondary reagent.

When the identity of the ligand is discovered, then antibodies that bindto the ligand can also be labeled and used to identify T-ALL cells.

Other embodiments provide a method of assessing whether a T-ALL patientis responsive to treatment by determining the number of T cells that aredetectably labeled in a patient sample prior to treatment and aftertreatment, and then determining that the patient is responding totreatment if the number of delectably labeled T-cells in thepre-treatment sample is significantly higher than the number ofdelectably labeled T-cells in the post-treatment sample.

ILT3 is Constitutively Expressed on Malignant Leukemia AML Cells ofMonocytic Differentiation

The results described herein regarding AML, show that ILT3 is a highlyspecific and sensitive biomarker that is constitutively expressed bynormal and leukemic myeloid precursors. Thus, ILT3 can be used as adiagnostic tool for distinguishing AML m4/m5 from other types of AML.

The key results show:

-   -   Flow cytometric analysis of AML samples (including AML with and        without monocytic differentiation) from patients with        non-involved bone marrow indicated that 80±9% of the CD14+        monocytes were ILT3+. Granulocytes were essentially negative.    -   No significant difference was found between the frequency of        ILT3+ precursor cells identified in bone marrow samples obtained        from patients previously treated for AML who are currently        disease free (N=13) and from non-AML controls (N=7).    -   Flow cytometric analysis of samples from patients with AML with        or without monocytic differentiation showed that ILT3 was        variably expressed by the leukemic cells (range 1-99%; mean±STD,        44.±41%).    -   The frequency of ILT3+ cells in patients with AML        m4/m5(AML/m4/m5) was dramatically elevated compared to patients        with AML that does not have monocytic differentiation        (AML/m1m2m3) Specifically 65±33% of the cells in AML/m4/m5 were        ILT3+, versus 1±1% in AML/m1/m2/m3 (p<0.0001). This means that        essentially all of the ILT3+ cells in AML/m4/m5 samples fell        above one standard deviation above the mean for AML/m1/m2/m3        samples. Based on this, a threshold of 10 to 15% was chosen,        such that if more than about 10-15% of the cells in the sample        are ILT3+, the sample is identified as AML/m4/m5 (ILT3+>10%;        p<0.0001); if less than about 10% to 15% or less of the cells in        the sample are ILT3+, the sample is not identified as AML/m4/m5.        Using this threshold, it was discovered that all of the 18 cases        so determined to be AML/m4/m5 agree with those 18 cases that        were previously identified as AML/m4/m5 analyzed using        established morphological, immunohistochemical and cytogenetic        criteria to determine the AML type.    -   CD11c, CD33, and HLA-DR were positive in >90% of the malignant        leukemic cells from patients having AML M4/M5. By contrast,        CD11c and CD33 were positive in >50% and 100%, respectively, of        leukemic cells from patients with AML M 1/M2 and M3. HLA-DR was        positive in >80% of patients with AML MI/M2.    -   CD14 was only expressed in 2 of 18 cases (61%) of AML m4/m5.    -   Co-expression of ILT3 and CD117 was observed in 50% of cases        with AML m4/m5, while co-expression of ILT3 and CD34 was 39%.    -   Flow cytometric results obtained after allogeneic stem cell        transplantation in a patient treated for AML m4/m5 indicated        ILT3+ staining and highlighted a population of monocytic        precursors that accounted for 64% of the CD45dim/low SSC cells        (4% of all nucleated cells).    -   ILT3+ AML M4/M5 cells co-expressed CD4, CD11c, CD33, CD34, CD64,        CD117, and HLA-DR, but they were negative for CD14, a phenotype        consistent with that of the original leukemia.    -   Cytogenetic data indicate that 27 of 32 patients with AML M4/M5        carried chromosomal abnormalities including a 5q deletion,        monosomies of chromosomes 5 and/or 7, and complex karyotypes (>3        unrelated abnormalities). One or more abnormalities were        detected in 5 of 17 patients with AML that does not have        monocytic differentiation (M1, M2, M3). None were detected in        any of the patients with ILT3+ AML M4/M5 (p=0.046).    -   Most of the cytogenetic abnormalities associated with a        favorable prognosis were found in the AML M3 (APL) group. Seven        out of 10 patients from this group carried the t(15, 17)        abnormality.    -   Cytogenetic abnormalities previously associated with monocytic        differentiation, namely t(9, 11) and 16q22, were observed in        only 3 of 14 patients with AML M4/M5.    -   Cytogenetic abnormalities previously associated with monocytic        differentiation, namely t(9, 11) and 16q22, were observed in        only 3 of 14 patients with AML M4/M5.

Construction of Human ILT3

Methods for making, isolating and purifying human ILT3, the soluble ILT3extracellular domain and other fragments of ILT3, or for making ILT3Fcusing, for example, recombinant technology or chemical synthesis, aredescribed in detail in Cosman, U.S. Pat. No. 6,448,035. Cosman alsodescribes variants, homologs and analogs of the ILT3 extracellulardomain and methods for making, identifying and isolating anti-ILT3antibodies.

Purified polypeptides comprising ILT3 or the extracellular domain ofhuman ILT3 can be used as the ILT3 ligand-binding probe in embodimentsof the present invention because this domain is known to contain theILT3 ligand-binding site. The polypeptides may be purified fromrecombinant expression systems (by subcloning the nucleic acid encodingthe ILT3 extracellular domain into an expression vector) or fromnaturally occurring cells. In an embodiment, the purification processesare such that no protein bands corresponding to proteins other than thedesired protein are detectable by SDS-polyacrylamide gel electrophoresis(SDS-PAGE).

A variety of methods for labeling the ILT3 ligand-binding probe and theILT3 binding probe (such as an anti-ILT3 mAb) are well known in the art,and include radioactive isotopes such as ¹²⁵I, ³²p, ³⁵S, and ³H,fluorophores, chemiluminescent agents, and enzymes, and also indirectlabeling with antibodies or other secondary reagent that targets andselectively binds to the ILT3 due to the high affinity of the antibodyfor ILT3. The choice of label depends on the sensitivity required, easeof conjugation with the primer, stability requirements, and availableinstrumentation. Methods for labeling polypeptides are well known in theart. See, e.g., Ausubel et al., 1992, hereby incorporated by reference.The probes described herein can be biotinylated and detected usinglabeled anti-biotin antibodies or probe can be detected usingavidin/streptavidin-tagged detection strategies such as enzyme reporters(e.g., horseradish peroxidase, alkaline phosphatase) in addition to thedescribed fluorescent probes.

Antibodies

The anti-ILT3 antibody can be a polyclonal or a monoclonal antibody. Theanti-ILT3 antibody is preferably a humanized antibody. In one embodimentof the methods described herein, the anti-ILT3 antibody is a fully humanantibody, mono- or polyclonal. The antibodies described herein may benon-cytolytic antibodies, such as in those embodiments where anti-ILT3is used as a secondary reagent that binds to ILT3 that is bound to ILT3ligand on a cancer cell.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a polypeptide ofthe invention, or against derivatives, fragments, analogs homologs ororthologs thereof. See, for example, ANTIBODIES: A LABORATORY MANUAL,Harlow and Lane (1988) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. Some of these antibodies are discussed below. Methods formaking fully human monoclonal antibodies are described in CURRENTPROTOCOLS IN IMMUNOLOGY, Ed. John E Coligan, Barbara E Bierer, David HMargulies, Ethan Shevach, Warren Strober. 1994-2006 John Wiley & Sons,Inc.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen-binding site capable ofimmunoreactions with a particular epitope of the antigen characterizedby a unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature, 256:495. In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The human or humanized anti-ILT3 antibodies used in embodiments used inembodiments of the invention are suitable for administration to humanswithout engendering an immune response by the human against theadministered immunoglobulin. Humanized forms of antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)that are principally comprised of the sequence of a humanimmunoglobulin, and contain minimal sequence derived from a non-humanimmunoglobulin. Humanization can be performed following the method ofWinter and co-workers (Jones et al. (1986) Nature, 321:522-525;Riechmann et al. (1988) Nature, 332:323-327; Verhoeyen et al. (1988)Science, 239:1534-1536), by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. (See also U.S. Pat.No. 5,225,539.)

Where an “antibody” is referred to herein with respect to the invention,it is normally understood that an antigen-binding portion thereof mayalso be used. An antigen-binding portion competes with the intactantibody for specific binding to the antigen, which is ILT3, ILT3Fc orthe ED. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entiretyfor all purposes). Antigen-binding portions may also be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. In some embodiments, antigen-binding portions includeFab, Fab′, F(ab′)₂, Fd, Fv, dAb, and complementarity determining region(CDR) fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of anantibody that is sufficient to confer specific antigen-binding to thepolypeptide.

As used herein, a Fd fragment means an antibody fragment that consistsof the V_(H) and C_(H1) domains; an Fv fragment consists of the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546 (1989)) consists of a V_(H) domain. Insome embodiments, the antibody is a single-chain antibody (scFv) inwhich a V_(L) and V_(H) domains are paired to form a monovalentmolecules via a synthetic linker that enables them to be made as asingle protein chain. (Bird et al., Science 242:423-426 (1988) andHuston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988).) In someembodiments, the antibodies are diabodies, i.e., are bivalent antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating twoantigen-binding sites. (See e.g., Holliger P. et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993), and Poljak R. J. et al., Structure2:1121-1123 (1994).) In some embodiments, one or more CDRs from anantibody of the invention may be incorporated into a molecule eithercovalently or noncovalently to make it an immunoadhesin thatspecifically binds to c-Met. In such embodiments, the CDR(s) may beincorporated as part of a larger polypeptide chain, may be covalentlylinked to another polypeptide chain, or may be incorporatednoncovalently.

An anti-ILT3 antibody or antigen-binding portion thereof can bederivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibodies or portion thereof are derivatizedsuch that the ILT3 binding is not affected adversely by thederivatization or labeling. Accordingly, the antibodies and antibodyportions of the invention are intended to include both intact andmodified forms of the human anti-ILT3 antibodies described herein. Forexample, an antibody or antibody portion of the invention can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (e.g., a bispecific antibody or a diabody), adetection agent (such as a fluorophore or radioactive label in thediagnostic embodiments described herein), a pharmaceutical agent (suchas a cytotoxic agent in order to kill the targeted cell expressing ILT3ligand), and/or a protein or peptide that can mediate association of theantibody or antibody portion with another molecule (such as astreptavidin core region or a polyhistidine tag).

The anti-ILT3 antibodies or an antigen-binding portion thereof cancomprise a label which may be a radioisotope or a particle which emitsradioactive radiation. This particle may be a radioactive element in aform which can be linked to the antibody or fragment thereof, preferablyin the form of a complex. For example an mAb labeled with ¹¹¹Indium maybe used to detect the target ILT3, and can also be used to kill targetedILT3-expressing cancer cells if the radiation emitted is strong enoughto be cytotoxic. Other suitable radioactive elements like ³⁵S or ¹³¹Ican be used. In embodiments where the particle has enough radiation tokill the targeted cancer cell, an anti-ILT3 antibody so labeled can beadministered in therapeutically effective amounts to treat a subjecthaving a cancer expressing ILT3 on its surface. Killing targetedILT3-expressing cancers or cancer cells expressing ILT3 ligand using theT-cell coreceptor CD3 (scDb EDGCD3) is described in detail in HoffmannP, Int J Cancer. 2005 May 20; 115(1):98-104, Serial killing of tumorcells by cytotoxic T cells redirected with a CD19-/CD3-bispecificsingle-chain antibody construct. In some embodiments the T cellcoreceptor CD3 is bound to the antibody to facilitate killing bycytotoxic T cells.

Another type of derivatized antibody is a labeled antibody that can beused to detect or visualize the antibody after it binds to the targetedantigen. Useful detection agents with which an antibody orantigen-binding portion of the invention may be derivatized includefluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors and the like. An antibodycan also be labeled with enzymes that are useful for detection, such ashorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase, glucose oxidase and the like. When an antibody is labeledwith a detectable enzyme, it is detected by adding additional reagentsthat the enzyme uses to produce a reaction product that can bediscerned. For example, when the agent horseradish peroxidase ispresent, the addition of hydrogen peroxide and diaminobenzidine leads toa colored reaction product, which is detectable. An antibody can also belabeled with biotin, and detected through indirect measurement of avidinor streptavidin binding. An antibody can also be labeled with apredetermined polypeptide epitope recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). In some embodiments,labels are attached by spacer arms of various lengths to reducepotential steric hindrance.

Alternatively, the antibodies or antigen-binding portion thereof may beused to make an immunotoxin, which comprises a cytotoxic agent.Immunotoxins are human-made proteins that consist of a targeting portionlike an antibody or fragment of one, linked to a toxin such as a celltoxic substance selected from the group comprising toxins, for exampleTaxol®, cytocalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etopside, tenopside, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy antracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosteron, glycocorticoids,procain, tetracaine, lidokaine, propranolol, puromycin, any bacterialtoxins including but not limited to Pseudomonas exotoxin PE38.

Anti-ILT3 monoclonal antibodies can be targeted against malignant cellsby several mechanisms: including Radioimmunotherapy (RIT) involves theuse of radioactively conjugated antibodies against cellular antigens. Insome cases the isotope is radionuclide iodine-131 that emits both betaand gamma radiation and decays with a half-life of 8 days. Lymphomas areclosely related to lymphoid leukemias like T-ALL and AML, which alsooriginate in lymphocytes but typically involve only circulating bloodand the bone marrow and do not usually form static tumors. Lymphomas arehighly radio-sensitive malignancies, and it is expected that leukemiaswill also be radio-sensitive. To limit radiation exposure, in anembodiment murine antibodies or humanized murine antibodies may beuseful for treatment with immunotoxins, as their higher immunogenicitypromotes rapid clearance from the body compared to fully humanantibodies.

Antibody-directed enzyme prodrug therapy (ADEPT) involves theapplication of cancer associated monoclonal antibodies which are linkedto a drug-activating enzyme. Subsequent systemic administration of anon-toxic agent results in its conversion to a toxic drug, and resultingin a cytotoxic effect which can be targeted at malignant cells. FrancisR J, Sharma S K, Springer C, et al. (2002). “A phase I trial of antibodydirected enzyme prodrug therapy (ADEPT) in patients with advancedcolorectal carcinoma or other CEA producing tumors”. Br J Cancer 87 (6):600-7.

Immunoliposomes are antibody-conjugated liposomes. Liposomes can carrydrugs or therapeutic nucleotides and when conjugated with monoclonal (orpolyclonal) antibodies, may be directed against malignant cells.Although this technique is still in its infancy, significant advanceshave been made. Immunoliposomes are particularly useful against bloodcancers because of easy contact with the targeted cancer cells.

The antibodies and antigen-binding portions of the present invention canbe incorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody or antigen-binding portion of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” means any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Someexamples of pharmaceutically acceptable carriers are water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Additionalexamples of pharmaceutically acceptable substances are wetting agents orminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody.

The new anti-ILT3 monoclonal antibodies A, B, C, and D or a portionthereof comprising the ILT3 ligand-binding site may be used for thepreparation of a medicament for the treatment of cancer that expressesILT3 ligand such as T-ALL. An anti-ILT3 antibody can also be derivatizedwith a chemical group such as polyethylene glycol (PEG), a methyl orethyl group, or a carbohydrate group. These groups are useful to improvethe biological characteristics of the antibody, e.g., to increase serumhalf-life.

IV. EXAMPLES Example 1

Flow Cytometry Method for Assaying the Expression of ILT3L UsingUnlabeled ILT3Fc and a Labeled Secondary Reagent with Specific Affinityfor ILT3Fc.

Bone marrow aspirate including peripheral blood mononuclear cells frompatients with T-ALL or cultured T-ALL cell lines were incubated with 10μg of unlabeled ILT3Fc for 30 minutes at 4° C. in 1000 of stainingbuffer. The staining buffer consisted of Tris buffered saline (TBS), 1-3mM Mn², and 1% BSA or a similar formulation. Of note, ILT3Fc ispreferably dissolved in a buffer compatible with the staining bufferused. Cells were washed three times in staining buffer. An appropriatenegative control consisted of the same type of cells incubated withbuffer alone (no ILT3Fc) or 10 μg human IgG during the first step.

The cells were then incubated with a secondary reagent (such asanti-Human ILT3 antibody) that binds to the ILT3 in the probe, whereinthe secondary reagent is conjugated to a fluorophore such as PC5) for(30 minutes, 4° C.) and then washed three more times in staining buffer.Any antibody (or biologically active fragment or variant thereof) thatselectively binds to ILT3 can be used as a secondary reagent.Commercially available monoclonal anti-human ILT3 monoclonal antibodieswere used. Monoclonal anti-human ILT3 antibodies (designated A, B, C andD) that were made in the lab by immunizing mice with ILT3Fc were alsotested (details for making anti-human ILT3 mAb are set forth in Example3). Commercially available anti-ILT3 antibodies from R&D Systemsinclude:

Human ILT3/CD85k Affinity FC AF2425 100 ug Purified Polyclonal Ab, GoatIgG Human ILT3/CD85k FC FAB24251A 100 Tests Allophycocyanin MAb (Clone293623), Mouse IgG2A Human ILT3/CD85k Biotinylated FC BAF2425 50 ugAffinity Purified PAb, Goat IgG Human ILT3/CD85k Fluorescein FCFAB24251F 100 Tests MAb (Clone 293623), Mouse IgG2A Human ILT3/CD85k MAb(Clone MAB2425 100 ug 293622), Mouse IgG2A Human ILT3/CD85k MAb (CloneFC MAB24251 100 ug 293623), Mouse IgG2A Human ILT3/CD85k PhycoerythrinFC FAB24251P MAb (Clone 293623), Mouse IgG2A *FC in the table abovemeans flow cytometry tested.

Also available is the antibody from Beckman Coulter IOTest® CD85k(ILT3)-PC5 PN IM3579; Item No: A46529.

A flow cytometry instrument and the appropriate acquisition/analysissoftware, such as BD Biosciences' FACSCalibur and Cell quest software,were used to analyze the ILT3Fc binding shown in FIGS. 1-4. T-ALL celllines were expanded and maintained in culture. (FIG. 2 (A)-(B)). Gatingidentifies leukemic cells in bone marrow aspirate from a patient withT-ALL are shown in FIG. 1 (A)-(B). On the other hand, normal T cellsfrom healthy individuals expressed low or no ILT3L (FIGS. 3 (A)-(B)-4(A)-(B)). CD4-gated T helper cells form a healthy blood donor expressed1% ILT3Fc binding (FIG. 3 (A)-(B)). CD3-gated T cells from a healthyblood donor also expressed low ILT3Fc binding at 12%. (FIG. 4 (A)-(B)).

The data generated by flow-cytometers can be plotted in a singledimension, to produce a histogram, or in two-dimensional dot plots oreven in three dimensions. The regions on these plots can be sequentiallyseparated, based on fluorescence intensity, by creating a series ofsubset extractions, termed “gates” or “gating.”

The number of labeled cells, and optionally also the amount of label percell, can be determined in different ways that depend on the reportermolecule. A preferred embodiment is using a fluorescent label and flowcytometry that can determine both the number of T-cells in a sample andamount of label per T-cell. Flow cytometry allows for the counting andexamining of cells by suspending them in a stream of fluid and passingthem by an electronic detection apparatus. This technique allows for thesimultaneous analysis of the physical and/or chemical characteristics ofup to thousands of particles per second in real time. Data accumulatedusing the flow cytometer can be analyzed using commercially availablesoftware.

Example 2

Targeted Lysis of T-ALL Cells that Express ILT3L with ILT3Ligand-Binding Probes

T-ALL cells were suspended in RPMI 1640 medium at a concentration of2×10⁶ cells/ml. First, cells were labeled with carbofluorescin diacetate(CFDA, 5 μM) for 10 minutes at 25° C., followed by thorough washing. Thecells were then incubated with ILT3Fc (an ILT3 ligand-binding probe)(various concentrations, 0-250 μg/ml) for one hour at room temperature,then washed three times and incubated with rabbit complement for anadditional hour.

Negative controls: the incubations were conducted without ILT3Fc,complement or both. Positive controls consisted of cells incubated withpooled human serum from allosensitized patients (containing anti HLAcytotoxic antibodies) as a first step and rabbit complement as a secondstep.

Finally, a viability dye (Ethidium Bromide, EtBr) was added and thecells were analyzed under a 488 nm light source. (Under 488 nm light,live cells appear green [CFDA+EtBr−] and dead cells appear red/orange[CFDA+EtBr+].)

% viability=(live cells/total cells)*100%

cytotoxicity=(dead cells/total cells)*100

The method can be varied using other molecules that selectively bind tothe targeted ILT3 ligand, incubating the cells with ILT3 ligand-bindingprobes that are not labeled, and then using a secondary reagent such asa labeled anti-ILT3 monoclonal antibody to detect the probe. Thecytolytic agent may be an entity other than rabbit complement, i.e.complement from a different species, dyphteria toxin (DT), etc.Anti-human globulin (AHG) can be added to the reaction to enhancecomplement cytotoxicity. The readout can be accomplished by variousinstruments such as a flow cytometer or a microscope (equipped with theappropriate lasers/light sources to detect the live/dead labelsemployed.)

In the experimental conditions described above, two T-ALL cell lineswere lysed (over 96-100% cytotoxicity) at all concentrations of ILT3Fcused (10-250 ug/ml). *Note that the binding of ILT3Fc itself did notlyse the cells; rather the binding of complement to the ligand wasresponsible for lysis.

Example 3

Methods for Making Anti-Human ILT3Fc Monoclonal Antibodies (mAb)

The procedures for production of monoclonal antibody were adapted fromCurrent Protocols in Immunology (1995) 2.5 contributed by Wayne M.Yokoyama. A person of skill in the art would know how to make mAb fromthe antigen.

Immunization to Produce Monoclonal Antibodies.

1. Mix equal volume of human ILT3Fc protein (1 mg/ml in PBS) and CFA(Complete Freunds adjuvant, Sigma) or IFA (Incomplete Freund's adjuvant,Sigma) in a 3-way stopcock until the two parts are completelyemulsified. The emulsion with CFA is used for the first timeimmunization. IFA emulsion is used for the rest of the immunization andboosting.

2. Inject 0.2 ml of emulsion (100 μg of ILT3Fc protein)intraperitoneally into female BALB/cj mice of 6-7 week old (JAX MICE) onday 1, 8, 15. The immunized mice are rested for 21 days, and are thenboosted with 0.2 ml emulsion with IFA. Three days after boosting, miceare sacrificed, and their spleens are taken for fusion.

Cell Fusion

1. One week before fusion, one vial of SP2/0-Ag14 myeloma cell line(drug-marked, nonsecretory; ATCC #CRL 1581) was cultured in completeDMEM medium (DMEM with 4500 mg glucose/L, 2 mM L-glutamine, 50 μg/mlgentamicin and 10% FBS, Sigma) at a cell density of no more than 10⁶/ml.One day before fusion, split the cells.

2. Spleens from the immunized mice were made into single-cellsuspensions by squeezing with angled forceps. Debris was removed bypassage through a fine-mesh metal screen. Transfer spleen cellsuspension to a sterile 50-ml conical centrifuge tube and fill withserum-free DMEM. Centrifuge 5 min at 1500 rpm (500×g), room temperature,and discard supernatant. Lyse red blood cells (RBC) by resuspendingpellet in 5 ml ammonium chloride solution. Let stand 5 min at roomtemperature. Add 45 ml sterile complete serum-free DMEM, and centrifugeas before. Resuspend cell pellet in serum-free medium, and wash thecells twice. Count the cells.

3. Separately harvest the SP2/0-Ag14 myeloma cells by transferring thecells to 50-ml conical centrifuge tubes. Wash myeloma cells three timeswith serum free DMEM medium. Count the cells.

4. Mix SP2/0-Ag14 myeloma and spleen cells from a whole spleen at a 1:10ratio in a 50-ml conical centrifuge tube. Spin down the mixture at 500 gfor 5 min and discard the supernatant. Perform the cell fusion at 37° C.by placing the tube containing the mixed-cell pellet in one of thedouble-beaker water baths in the laminar flow hood. Using a 1-ml pipet,add 1 ml pre-warmed 50% PEG (Sigma) to the mixed-cell pelletdrop-by-drop over 1 min, stirring the cells with the pipet tip aftereach drop. Stir for an additional minute. Using a clean pipet, add 1 mlpre-warmed serum-free DMEM to the cell mixture drop-by-drop over 1 min,stirring after each drop. Repeat once with an additional 1 ml ofpre-warmed complete serum-free DMEM. With a 10-ml pipet, add 7 mlpre-warmed serum-free DMEM drop-by-drop over 3 min. Centrifuge 5 min at500×g, room temperature, and discard the supernatant. With a clean 10-mlpipet, forcefully discharge 10 ml prewarmed complete DMEM to the cellpellet. Repeat the step with 10 ml prewarmed complete DMEM medium untiltotal volume of 30 ml is reached. If necessary, allow clumps to settleand disrupt with the pipet tip. Gently aspirate 10 ml of cell suspensionwith a 10-ml pipet. Add 2 drops (100 to 125 μl) of suspension to eachwell of a 96-well flat-bottom plate (Fisher Scientific); continue untilentire suspension is plated. Incubate overnight in a humidified 37° C.,5% CO2 incubator.

4. After one day of incubation (day 2), add 2 drops of 1×HAT (Sigma) incomplete DMEM-medium to each well with a 10-ml pipet. Replace halfvolume of medium in each well with 1×HAT medium on day 3, 4, 5, 6, 8,and 10. On day 15, replace the medium with 1×HT (Sigma) medium. Afterreplacing medium with HT medium twice, feed the cells with complete DMEMmedium.

Screening Primary Hybridoma Supernatants

1. Check cell growth under microscope. Screening should be performedwhen the growing cells occupy 10-25% space of a well.

2. Coat the immulon microplate (Fisher Scientific) with ILT3Fc andcontrol human Ig (Sigma). Block the plates with PBS containing 0.05%Tween 20 and 0.25% BSA after coating.

3. Add the supernatant from the selected wells to ILT3Fc and Ig coatedwells. Incubate the plate for 1 hour, wash 3 times. Add goat anti-mouseperoxidase antibody (Sigma). Incubate for 1 hour, and then wash 3 times.Adding the substrate prepared from peroxidase substrate tablets (Sigma).Pick up the wells which are positive for ILT3Fc but negative for humanIg. All the procedures of ELISA are performed according tomanufacturer's instructions.

Subcloning the Hybridoma Cells which Produce ILT3Fc Positive but HumanIg Negative Antibody.

1. Take a spleen from normal BALB/c female mouse; prepare single spleencells as described in cell fusion.

2. Add 50 μl of complete DMEM medium containing 10⁵ spleen cells to eachwell of a 96-well flat-bottom plate.

3. Take the cells from the wells which have the hybridoma cellsproducing anti-ILT3Fc antibodies. Add 50 μl of complete DMEM mediumcontaining average 0.5 or 1 cell/well to each well of a 96-wellflat-bottom plate containing spleen cells.

4. Check the plates for cell growth. Add or exchange medium asnecessary. Once the hybridoma cells reach 10-25% space of a well, screenthe supernatant from that well as described.

5. Expand and freeze the cells from the clones producing ILT3Fc specificantibody.

6. To further confirm that those monoclonal antibodies are against ILT3molecule, use the supernatants from those clones to stain KG1.ILT3 cellsby indirect immunostaining and analyzed by flow.

Materials and Methods Patient Case Selection

A total of 61 consecutive specimens, which included 52 bone marrow and 9peripheral blood samples, were analyzed between Jan. 1, 2003 and Dec.31, 2006, according to a protocol approved by the IRB of ColumbiaUniversity. Samples were obtained for diagnostic purposes from a totalof 56 patients followed at our institution. ILT3 expression was testedon left-over samples after the diagnostic work-up has been completed.Diagnosis was established based on morphology, immunohistochemistry,flow cytometry, and cytogenetic analysis. Out of 61 specimens, 32 bonemarrow and 9 peripheral blood samples were obtained from patients withAML (N=37). For the purpose of this study, leukemia cases werecategorized according to the FAB criteria (Bennett et al., 1985). Casesof FAB M4, M4Eo, M5a and M5b were considered as displaying monocyticdifferentiation, while FAB MO, M1, M2, M3 (acute promyelocyticleukemia), M6 and M7 were not. The remaining 20 samples were obtainedfrom patients with no evidence of bone marrow involvement, asestablished by clinical evaluation and morphology, flow cytometry andcytogenetic analysis. Thirteen of the non-involved samples were obtainedfrom patients previously treated for AML (3 AML without differentiation,5 APL, 3 AML with monocytic differentiation, 2 undefined AML types),while the remaining 7 samples were obtained from patients with otherdiseases (I aplastic anemia, 2 gastric B-cell lymphoma, 2 acutelymphocytic leukemia in remission, and 2 viral infections).

Clinical Parameters

The following clinical parameters were analyzed in relation to ILT3expression by leukemic cells in patients with AML: age, gender, AMLtype, WBC, peripheral blood monocyte count. hemoglobin level, plateletcount, frequency of blasts in peripheral blood and bone marrow,chromosomal abnormalities and overall survival.

Flow Cytometry

Immunophenotypic characterization of bone marrow and peripheral bloodsamples was performed using three-color flow cytometry, as previouslydescribed (57). The following monoclonal antibodies were used:anti-CD45, -CD4, -Co11e, -CD13, -CD14, -CD16, -CD33, CD34, -CD45, -CD56,-CD64, -CD117, -HLA-DR-MPO (BD BioScience, San Jose, Calif.), and ILT3(Beckman Coulter, Miami, Fla.). Leukemic cells were considered positivefor any given marker if >10% of the cells expressed that marker. Allsamples were tested within 48 h of collection. Cells were run andanalyzed on a FACSCalibur (BD Biosciences) using CellQuestPro software.

Statistical Analysis

The relationship between ILT3 expression and clinical parameters wasstudied using multiple regression analysis, Student's t test ofsignificance and Chi-square test (ffiM SPSS Statistics 17.0). KaplanMeier analysis was used to assess patient survival in ILT3+ and ILT3−AMLgroups.

Results

A. ILT3 is a Highly Specific and Sensitive Marker that can ReliablyDistinguish AML m4/m5 from Other Types of AML

To characterize the expression of inhibitory receptor ILT3 on normal andneoplastic hematopoietic precursors, we analyzed 20 bone marrow samplesobtained from patients with no evidence of neoplastic disease and 41specimens obtained from patients with AML. Flow cytometric analysis ofnon-involved bone marrow indicated that a high proportion of the CD14+monocytes, specifically 80±9%, expressed ILT3 (FIG. 5), whilegranulocytes were essentially negative. This profile is similar to thatpreviously reported for peripheral blood myelomonocytic cells (47),(57). Within the CD45dim/low side scatter (SSC) gate, which includeshematopoietic precursors, 10±8% of the gated cells (0.4±0.3% of allnucleated cells) expressed ILT3. There was no significant differencebetween the frequency of ILT3+ cells identified in the bone marrowsamples obtained from patients previously treated for AML, who arecurrently disease free (N=13), and the frequency of ILT3+ precursorcells from non-AML patients (N=7). ILT3 precursors co-expressed CD33 andone or more of the following markers: CD34, CD117 (c-kit), CD13, CO11c,and CD14.

Co-expression of ILT3 and B cell markers was not observed, indicatingthat B cell precursors do not express ILT3 (data not shown). Asillustrated in FIG. 5, ILT3 positive cells included CD14+ and CD 14−cells, consistent with a maturation pattern in which ILT3 expression bybone marrow monocytic cells is acquired prior to CD14 expression.Co-expression of ILT3 and the early markers CD34 and CD 117 suggeststhat ILT3 is acquired at an early step of hematopoietic differentiation.

In patients with AML, leukemic cells represented 22-93% (mean±STD,55±33%) of the bone marrow nucleated cells (N=32) or peripheral whiteblood cells (N=9), as determined by flow cytometry. The diagnosis of AMLwas confirmed by morphological, immunohistochemical and cytogeneticfindings. Flow cytometric analysis indicated that ILT3 was variablyexpressed by the leukemic cells (range 1-99%; mean±STD, 44.±41%).However, the frequency of ILT3 positive cells in patients with AMLdisplaying monocytic differentiation (M4/M5) was significantly higherthan that observed in patients with other forms of AML, namely 65±33%versus 1±1% of the leukemic cells (p<0.0001) (Table 1). All of the 18cases of AML m4/m5 were determined to be positive for ILT3 (ILT3+>10-15%gene), while all cases with other types of AML were negative(ILT3+˜10-15%; p<0.0001; Table 1).

None of the other cellular markers was as sensitive or specific as ILT3in distinguishing AML m4/m5 from other forms of AML (FIG. 6). AlthoughCD11c, CD33 and HLA-DR were positive in >90% of the malignant cells frompatients having AML M4/M5, these markers lacked specificity as they wereonly expressed in a significant fraction of other AML types. CD11c andCD33 were positive in >50% and 100%, respectively, of patients with AMLM1/M2 and M3, while HLA-DR was positive in >80% of patients with AMLMI/M2. Of note, the monocytic marker CDI4 was expressed in only II outof 18 cases (61%) of AML displaying monocytic differentiation. Theseresults indicate that ILT3 is a highly specific and sensitive markerthat can reliably distinguish AML m4/m5 from other types of AML.

Notably, in patients with AML displaying monocytic differentiation, ILT3was expressed by leukemic cells at various stages of maturation. Asillustrated in FIG. 7 (panels A, B, C), ILT3 was co-expressed byCD34+/−CD117+CD14− monoblasts and promonocytes as well as by moredifferentiated CD34−CD 117−CD 14+/− leukemic cells. Overall,co-expression of ILT3 and CD 117 was observed in 50%, whileco-expression of ILT3 and CD34 was seen in 39% of cases with AMLdisplaying monocytic differentiation. (FIG. 6). As described above, ILT3was absent on leukemic cells from patients with AML lacking features ofmonocytic differentiation (FIG. 7 E, F).

B. Use of ILT3 as a Marker for Post-Treatment Monitoring of Patientswith AML

To determine whether ILT3 can be used for post-treatment monitoring ofpatients with AML displaying monocytic differentiation, we analyzed ILT3expression in bone marrow samples taken from patients who relapsed aftertreatment. FIG. 7(D) illustrates the flow cytometric results obtainedafter allogeneic stem cell transplantation in a patient treated for AMLwith monocytic differentiation. ILT3 staining highlighted a populationof monocytic precursors, which accounted for 64% of the CD45dim/low SSCcells (4% of all nucleated cells}. The frequency of ILT3+ cells withinthe precursor gate was significantly higher than that observed innon-involved bone marrow (FIG. 5), suggesting that the ILT3+ cells wereneoplastic. These cells co-expressed CD4, CD11c, CD33, CD34, CD64,CD117, and HLA-DR and were negative for CD 14, a phenotype consistentwith that of the original leukemia. Leukemia relapse was confirmed bycytogenetic analysis. These results indicate that ILT3 expression ismaintained after AML treatment and is a useful marker for diseasemonitoring in AML with mono differentiation.

C. Statistical Significance and Clinical Correlations

As indicated above, there was a statistically significant correlationbetween ILT3 expression and the AML type ILT3 was positive in all AMLM4/M5 cases yet in none of the M1/M2 and M3 cases (p<0.0001; Table I).Although the frequency of leukemic blasts present in the bone marrow didnot vary significantly in the ILT3+ versus ILT3− AML groups, thefrequency of circulating leukemic blasts was significantly lower in theILT3+ group compared to the ILT3− group (p<0.009; Table I). Monocytosis(an increase in the number of monocytes circulating in the blood)occurred more frequently in patients with ILT3+ AML compared to patientswith ILT3-AML, although the difference did not reach statisticalsignificance (p=0.08). There were no significant differences between thetwo groups for the following parameters: age, gender, WBC, hemoglobinlevels and platelet count.

Cytogenetic data were available for 32 patients with AML. Of these, 27carried chromosomal abnormalities. The following cytogeneticabnormalities associated with unfavorable outcome in patients with AMLwere detected in our study patients: 5q deletion, monosomies ofchromosomes 5 and/or 7, and complex karyotype (>3 unrelatedabnormalities).

One or more of these abnormalities were detected in 5 out of 17 patientswith ILT3− AML, but it was not detected in any of the patients withILT3+ AML (p=0.046; Table 1). All patients with adverse abnormalitieswere from the M0-M2 subgroup. Most of the cytogenetic abnormalitiesassociated with a favorable prognosis were found in the AML M3 (APL)group. Thus, 7 out of 10 patients from this group carried the t(15; 17)abnormality. Inv16, which has been also associated with favorableprognosis, was detected in only one patient from the ILT3+M4/M5 group.The most frequent abnormalities found in the ILT3+ AML group were thoseassociated with an intermediate prognosis (Table 1; p<0.001).Cytogenetic abnormalities previously associated with monocyticdifferentiation, namely t(9, 11) and 16q22, were observed in only 3 of14 patients with AML m4/m5. Using multiple regression analysis, the AMLtype and the presence of cytogenetic abnormalities associated withintermediate prognosis were the clinical parameters most significantlycorrelated with ILT3 expression in our patients.

Patient survival in relation to ILT3 express ion was analyzed using theKaplan-Meier analysis. As indicated in FIG. 8, patients from the ILT3negative/M3 subgroup had the best survival, while those from the ILT3positive/M4/M5 group tended to have the lowest survival.

Although the difference between groups did not reach statisticalsignificance, ILT3 expression appears to have an adverse effect onsurvival.

In the present specification, the invention has been described withreference to specific embodiments thereof. It will, however, be evidentthat various modifications and changes may be made thereto withoutdeparting from the broader spirit and scope of the invention. Thespecification and drawings are, accordingly, to be regarded in anillustrative rather than a restrictive sense. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference as if set forth herein in their entirety, except whereterminology is not consistent with the definitions herein. Althoughspecific terms are employed, they are used as in the art unlessotherwise indicated.

REFERENCES

The entire contents of the following references are hereby incorporatedby reference as if fully set forth herein, except for terminology thatis inconsistent with the terminology used herein.

-   1. Chang C C, Liu Z, Vlad G, Qin H, Qiao X, Mancini D M, Marboe C C,    Cortesini R, Suciu-Foca N. Ig like transcript 3 regulates expression    of proinflammatory cytokines and migration of activated T cells. J    Immunol. 1; 182(9):5208-16, 2009.-   2. Vlad G, D'Agati V D, Zhang Q Y, Liu Z, Ho E K, Mohanakumar T,    Hardy M A, Cortesini R, Suciu-Foca N. ILT3-Fc suppresses T cell    responses to allogeneic human islet transplants in Hu-NOD/SCID mice.    Diabetes 57(7):1878-86, 2008.-   3. Suciu-Foca N, Feirt N, Zhang Q Y, Vlad G, Liu Z, Lin H, Chang C    C, Ho E K, Colovai A I, Kaufman H, D'Agati V D, Thaker H M, Remotti    H, Galluzzo S, Cinti P, Rabitti, C, Allendorf A, Chabot J, Caricato    M, Coppola R, Berloco P, Cortesini R. Soluble Ig-Like Transcript 3    Inhibits Tumor Allograft Rejection in Hu-SCID Mice and T Cell    Responses in Cancer Patients. J Immunol. 178(11):7432-41, 2007.-   4. Kim-Schulze, Scotto L, Vlad G, Piazza F, Lin H, Liu Z, Cortesini    R, Suciu-Foca N. Recombinant Ig-Like Transcript 3-Fc Modulates T    Cell Responses via Induction of Th Anergy and Differentiation of    CD8+T Suppressor Cells. J Immunol. 176(5):2790-8, 2006.-   5. Kim-Schulze S, Seki T, Vlad G, Scotto L, Fan J, Colombo P C, Liu    J, Cortesini R, Suciu-Foca N. Regulation of ILT3 Gene Expression by    Processing of Precursor Transcripts in Human Endothelial Cells. Am J    Transpl, 6(1):76-82, 2005.-   6. Vlad G, Cortesini R, Suciu-Foca N. License to Heal: Bidirectional    Interaction of Antigen-Specific Regulatory T Cells and Tolerogenic    APC. J Immunol. 174(10): 5907-14, 2005.-   7. Manavalan J S, Kim-Schulze S, Scotto L, Naiyer A J, Vlad G,    Colombo P C,-   Marboe C, Mancini D, Cortesini R, Suciu-Foca N. Alloantigen specific    CD8+CD28−FOXP3+T suppressor cells induce ILT3+ILT4+ tolerogenic    endothelial cells inhibiting alloreactivity. Int Immunol.    16(8):1055-1068, 2004.-   8. Chang C C, Ciubotariu R, Cortesini R, Colonna M, Lederman S,    Dalla-Favera R, Suciu-Foca N. Tolerization of dendritic cells by    T(s) cells: the crucial role of inhibitory receptors ILT3 and ILT4.    Nat Immunol. 3(3):237-243, 2002.-   9. Ciubotariu R, Colovai A I, Pennesi G, Liu Z, Smith D, Berlocco P,    Cortesini R, Suciu-Foca N. Specific suppression of human CD4+Th cell    responses to pig MHC antigens by CD8+CD28-regulatory T cells. J.    Immunol. 161:5193-5202, 1998.-   10. Ciubotariu R, Liu Z, Colovai A I, Ho E, Itescu S, Ravalli S,    Hardy M A, Cortesini R, Rose E A, Suciu-Foca N. Persistent    allopeptide reactivity and epitope spreading in chronic rejection of    organ allografts. J. Clin. Invest. 101:398-405, 1998.-   11. Liu Z, Colovai A I, Tugulea S, Reed E F, Fisher P E, Mancini D,    Rose E A, Cortesini, R, Michler R E and Suciu-Foca N. Indirect    recognition of donor HLA-DR peptides in organ allograft    rejection. J. Clin. Invest. 98:1150-1157, 1996.-   12. Harris P E, Maffei A, Colovai A I, Kinne J, Tugulea S,    Suciu-Foca N. Predominant HLA-class II bound self peptides of a    hematopoietic progenitor cell line. Blood 87:5104-5112, 1996.-   13. Liu Z, Sun Y K, Xi Y P, Maffei A, Reed E, Harris P,    Suciu-Foca N. Contribution of direct and indirect recognition    pathway to T cell alloreactivity. J. Exp. Med. 177:1643-1650, 1993.-   14. Harris P E, Lupu F, Hong B, Reed E F, Suciu-Foca N.    Differentiation stage specific “self” peptides bound by MHC-class I    molecules. J. Exp. Med. 177:783-790, 1993.-   15. Liu Z, Sun Y K, Xi Y P, Harris P, Suciu-Foca N. T cell    recognition of self-human histocompatibility leucocyte antigens    (HLA)-DR peptides in context of syngeneic HLA-DR molecules. J. Exp.    Med. 175:1663-1668, 1992.-   16. Harris P E, Strba-Cechova K, Rubinstein P, Mann D, King D W,    Suciu-Foca N. Amplification of T cell blastogenic responses in    healthy individuals and patients with acquired immunodeficiency    syndrome. J. Clin. Invest. 85:746-756, 1990.-   17. Reed E, Hardy M, Benvenisty A, Lattes C, Brensilver J, McCabe R,    Reemstma K, King D W, Suciu-Foca N. Effect of anti-idiotypic    antibodies to HLA on graft survival in renal-allograft recipients.    New Engl. J. Med. 316:1450-1455, 1987.-   18. Sangster R N, Minowada, J, Suciu-Foca N, Minden M, Mak T W.    Rearrangement and expression of the α, β, and γ chain T cell    receptor genes in human thymic leukemia cells and functional T    cells. J. Exp. Med. 163:1491-1508, 1986.-   19. Suciu-Foca N, Reed E, Rubinstein P, MacKenzie W, Ng A, King D W.    A late differentiation antigen (LDA1) associated with the helper    inducer function of human T lymphocytes. Nature 318:465-467, 1985.-   20. Kronenberg M, Goverman J, Haars R, Malissen M, Kraig E, Phillips    L, Delovitch T, Suciu-Foca N, Hood L. Rearrangement and    transcription of the beta-chain genes of the T cell antigen receptor    in different types of murine lymphocytes. Nature 313:647-653, 1985.-   21. Toyonaga B, Yanagi Y, Suciu-Foca N, Mindem M, Mak T W.    Rearrangements of the T cell receptor gene YT35 in human DNA from    thymic leukemic T cell lines and functional helper, killer and    suppressor T cell clones. Nature. 311:385-387, 1984.-   22. Yoshikai Y, Yanagi Y, Suciu-Foca N, Mak T W. Presence of T cell    receptor mRNA in functionally distinct T cells and elevation during    intrathymic differentiation. Nature 310:506-508.1984.-   23. Suciu-Foca N, Rubinstein P, Popovic M, Gallo R C, King D W.    Reactivity of HTLV transformed human T cell lines to MHC Class-II    antigens. Nature 321:275-277, 1984.-   24. Hoffmann P, Int J Cancer. 2005 May 20; 115(1):98-104, Serial    killing of tumor cells by cytotoxic T cells redirected with a    CD19-/CD3-bispecific single-chain antibody construct.-   25. Swerdlow S H, Campo E, Harris N L, Jaffe E S, Pileri S A, Stein    H, Thiele J, Vardiman J W, editor. WHO classification of tumors of    hematopoietic and lymphoid tissues, Fourth edition. Lyon:    International Agency for Research and Cancer; 2008.-   26. Foran J M. New prognostic markers in acute myeloid leukemia:    perspective from the clinic. Hematology Am Soc Hematol Educ Program.    201 0; 201 0:47-55.-   27. Becker M W, Jordan C T. Leukemia stem cells in 2010: current    understanding and future directions. Blood Rev. 2011 March;    25(2):75-8 1.-   28. Estey E, Dohner H. Acute myeloid leukaemia. Lancet. 2006 Nov.    25; 368(9550): 1894-907.-   29. Marcucci G, Haferlach T, Dohner H. Molecular genetics of adult    acute myeloid leuke rrti a: prognostic and therapeutic implications.    J Clin Oncol. 2011 Feb. 10; 29(5):475-86.-   30. Schlenk R F, Dohner K, Krauter J, Frohling S, Corbacioglu A,    Bullinger L, et al. Mutations and treatment outcome in    cytogenetically normal acute myeloid leukemia. N Engl J Med. 2008    May 1; 358(18): 1909-1 8.-   31. Bacher U, Kern W, Schnittger S, Hiddemann W, Schoch C, Haferl    ach T. Further correlations of morphology according to FAB and WHO    classification to cytogenetics in de novo acute myeloid leukemia: a    study on 2,235 patients. Ann Hematol. 2005 November; 84(12):785-91.-   32. Byrd J C, Mrozek K, Dodge R K, Carroll A J, Edwards C G, Arthur    D C, et al. Pretreatment cytogenetic abnormalities are predictive of    induction success, cumulative incidence of relapse, and overall    survival in adult patients with de novo acute myeloid leukemia:    results from Cancer and Leukemia Group B (CALGB 8461). Blood. 2002    Dec. 15; 100(13):4325-36.-   33. Grimwade D, Hills R K, Moorman A V, Walker H, Chatters S,    Goldstone A H, et al. Refinement of cytogenetic classification in    acute myeloid leukemia: determination of prognostic significance of    rare recurring chromosomal abnormalities among 5876 younger adult    patients treated in the United Kingdom Medical Research Council    trials. Blood. 2010 Jul. 22; 11 6(3):354-65.-   34. Santamaria C M, Chillon M C, Garcia-Sanz R, Perez C, Caballero M    D, Ramos F, et al. Molecular stratification model for prognosis in    cytogenetically normal acute myeloid leukemia. Blood. 2009 Jul. 2;    114(1):148-52.-   35. Mardis E R, Ding L, Dooling D J, Larson D E, McLellan M D, Chen    K, et al. Recurring mutations found by sequencing an acute myeloid    leukemia genome. N Engl J Med. 2009 Sep. 10; 361(11):1058-66.-   36. Tallman M S. Relevance of pathologic classifications and    diagnosis of acute myeloid leukemia to clinical trials and clinical    practice. Cancer Treat Res. 2004; 121:45-67.-   37. Dohner H, Estey E H, Amadori S, Appelbaum F R, Buchner T,    Burnett A K, et al. Diagnosis and management of acute myeloid    leukemia in adults: recommendations from an international expert    panel, on behalf of the European LeukemiaNet. Blood. 2010 Jan. 21;    115(3):453-74.-   38. Appelbaum F R, Gundacker H, Head D R, Slovak M L, Willman C L,    Godwin J E, et al. Age and acute myeloid leukemia. Blood. 2006 May    1; 107(9):3481-5.-   39. Poll yea D A, Kohrt H E, Medeiros B C. Acute myeloid leukaemia    in the elderly: a review. Br J Haematol. 2011 March; 152(5):524-42.-   40. Klco J M, Kulkarni S, Kreisel F H, Nguyen T D, Hassan A, Frater    J L. Immunohistochemical analysis of monocytic leukemias: usefulness    of CD14 and Kruppel-like factor 4, a novel monocyte marker. Am J    Clin Pathol. 2011 May; 135(5):720-30.-   41. Manaloor E J, Neiman R S, Heilman D K, Albitar M, Casey T,    Vattuone T, et al. Immunohistochemistry can be used to subtype acute    myeloid leukemia in routinely processed bone marrow biopsy    specimens. Comparison with flow cytometry. Am J Clin Pathol. 2000    June; 113(6):814-22.-   42. Garcia C, Gardner D, Reichard K K. CD163: a specific    immunohistochemical marker for acute myeloid leukemia with monocytic    differentiation. Appl lmmunohistochem Mol Morphol. 2008 October;    16(5):417-21.-   43. Harms P W, Bandarchi B, MaL. CD163 expression in leukemia cutis.    J Cutan Pathol. 2010 September; 37(9):953-7.-   44. Dunphy C H, Tang W. The value of CD64 expression in    distinguishing acute myeloid leukemia with monocytic differentiation    from other subtypes of acute myeloid leukemia: a flow cytometric    analysis of 64 cases. Arch Pathol Lab Med. 2007 May; 131(5):748-54.-   45. Gorczyca W. Flow cytometry immunophenotypic characteristics of    monocytic population in acute monocytic leukemia (AML-M5), acute    myelomonocytic leukemia (AML-M4), and chronic myelomonocytic    leukemia (CMML). Methods Cell Bioi. 2004; 75:665-77.-   46. Dunphy C H, Orton S O, Mantell J. Relative contributions of    enzyme cytochemistry and flow cytometric immunophenotyping to the    evaluation of acute myeloid leukemias with a monocytic component and    of flow cytometric immunophenotyping to the evaluation of absolute    monocytoses. Am J Clin Pathol. 2004 December; 122(6):865-74.-   47. Cella M, Dohring C, Samaridis J, Des sing M, Brockhaus M,    Lanzavecchia A, et al. A novel inhibitory receptor (IL T3) expressed    on monocytes, macrophages, and dendritic cells involved in antigen    processing. J Exp Med. 1997 May 19; 185(10):1743-51.-   48. Kim-Schulze S, Seki T, Vlad G, Scotto L, Fan J, Colombo P C, et    al. Regulation of ILT3 gene expression by processing of precursor    transcripts in human endothelial cells. Am J Transplant. 2006    January; 6(1):76-82.-   49. Mori Y, Tsuji S, Inui M, Sakamoto Y, Endo S, Ito Y, et al.    Inhibitory immunoglobulinlike receptors LILRB and PIR-B negatively    regulate osteoclast development. J Immunol. 2008 Oct. 1;    181(7):4742-51.-   50. Ravetch J V, Lanier L L. Immune inhibitory receptors. Science.    2000 Oct. 6; 290(5489):84-9.-   51. Anderson K J, Allen R L. Regulation of T-cell immunity by    leucocyte immunoglobulinlike receptors: innate immune receptors for    self on antigen-presenting cells. Immunology. 2009 May; 127(1):8-17.-   52. Lu H K, Rentero C, Raftery M J, Borges L, Bryant K, Tedla N.    Leukocyte Ig-like receptor B4 (LILRB4) is a potent inhibitor of    FcgammaRI-mediated monocyte activation via dephosphorylation of    multiple kinases. J Bioi Chern. 2009 Dec. 11; 284(50):34839-48.-   53. Chang C C, Ciubotariu R, Manavalan J S, Yuan J, Colovai A I,    Piazza F, et al. Tolerization of dendritic cells by T(S) cells: the    crucial role of inhibitory receptors ILT3 and ILT4. Nat Immunol.    2002 March; 3(3):237-43.-   54. Chang C C, Liu Z, Vlad G, Qin H, Qiao X, Mancini D M, et al.    Ig-like transcript 3 regulates expression of proinflammatory    cytokines and migration of activated T cells. J Immunol. 2009 May 1;    182(9):5208-16.-   55. Chang C C, Vlad G, D'Agati V D, Liu Z, Zhang Q Y, Witkowski P,    et al. BCL6 is required for differentiation of Ig-like transcript    3-Fc-induced CD8+T suppressor cells. J Immunol. 2010 Nov. 15;    185(10):5714-22.-   56. Munitz A. Inhibitory receptors on myeloid cells: new targets for    therapy? Pharmacal Ther. 2010 January; 125(1):128-37.-   57. Colovai A I, Tsao L, Wang S, Lin H, Wang C, Seki T, et al.    Expression of inhibitory receptor ILT3 on neoplastic B cells is    associated with lymphoid tissue involvement in chronic lymphocytic    leukemia. Cytometry B Clin Cytom. 2007 September; 72(5):354-62.-   58. Marcucci G, Radmacher M D, Maharry K, Mrozek K, Ruppert A S,    Paschka P, et al. MicroRNA expression in cytogenetically normal    acute myeloid leukemia. N Engl J Med. 2008 May 1; 358(18):1919-28.-   59. Haferlach C, Mecucci C, Schnittger S, Kohlmann A, Mancini M,    Cuneo A, et al. AML with mutated NPM1 carrying a normal or aberrant    karyotype show overlapping biologic, pathologic, immunophenotypic,    and prognostic features. Blood. 2009 Oct. 1; 114(14):3024-32.-   60. Valk P J, Verhaak R G, Beijen M A, Erpelinck C A, Barjesteh van    Waalwijk van DoornKhosrovani S, Boer J M, et al. Prognostically    useful gene-expression profiles in acute myeloid leukemia. N Engl J    Med. 2004 Apr. 15; 350(16):1617-28.-   61. Lutherborrow M, Bryant A, Jayaswal V, Agapiou D, Palma C, Yang Y    H, et al. Expression profiling of cytogenetically normal acute    myeloid leukemia identifies microRNAs that target genes involved in    monocytic differentiation. Am J Hematol. 2011 January; 86(1):2-11.-   62. Spoo A C, Lubbert M, Wierda W G, Burger J A. CXCR4 is a    prognostic marker in acute myelogenous leukemia. Blood. 2007 Jan.    15; 109(2):786-91.-   63. Becker P S, Kopecky K J, Wilks A N, Chien S, Harlan J M, Willman    C L, et al. Very late antigen-4 function of myeloblasts correlates    with improved overall survival for patients with acute myeloid    leukemia. Blood. 2009 Jan. 22; 113(4):866-74.-   64. Bruggemann M, RaffT, Flohr T, Gokbuget N, Nakao M, Droese J, et    al. Clinical significance of minimal residual disease quantification    in adult patients with standard-risk acute lymphoblastic leukemia.    Blood. 2006 Feb. 1; 107(3):1116-23.-   65. Kronke J, Schlenk R F, Jensen K O, Tschurtz F, Corbacioglu A,    Gaidzik V I, et al. Monitoring of minimal residual disease in    NPM1-mutated acute myeloid leukemia: a study from the    German-Austrian acute myeloid leukemia study group. J Clio Oncol.    2011 Jul. 1; 29(19):2709-16.-   66. San Miguel J F, Vidriales M B, Lopez-Berges C, Diaz-Mediavilla    J, Gutierrez N, Canizo C, et al. Early immunophenotypical evaluation    of minimal residual disease in acute myeloid leukemia identifies    different patient risk groups and may contribute to postinduction    treatment stratification. Blood. 2001 Sep. 15; 98(6):1746-51.-   67. Sioud M, Floisand Y. TLR agonists induce the differentiation of    human bone marrow CD34+ progenitors into COlle+CD80/86+DC capable of    inducing a Th1-type response. Eur J Immunol. 2007 October; 37(1    0):2834-46.-   68. De Luca K, Frances-Duvert V, Asensio M J, Ihsani R, Debien E,    Taillardet M, et al. The TLR1/2 agonist PAM(3)CSK(4) instructs    commitment of human hematopoietic stem cells to a myeloid cell fate.    Leukemia. 2009 November; 23(11):2063-74.-   69. Esplin B L, Shimazu T, Weiner R S, Garrett K P, Nie L, Zhang Q,    et al. Chronic exposure to a TLR ligand injures hematopoietic stem    cells. J Immunol. 2011 May 1; 186(9):5367-75.-   70. Ujike A, Takeda K, Nakamura A, Ebihara S, Akiyama K, Takai T.    Impaired dendritic cell maturation and increased T(H)2 responses in    PIR-B(−/−) mice. Nat Immunol. 2002 June; 3(6):542-8.-   71. Geissmann F, Manz M G, Jung S, Sieweke M H, Merad M, Ley K.    Development of monocytes, macrophages, and dendritic cells. Science.    2010 Feb. 5; 327(5966):656-61.-   72. Fogg D K, Sibon C, Miled C, Jung S, Aucouturier P, Littman D R,    et al. A clonogenic bone marrow progenitor specific for macrophages    and dendritic cells. Science. 2006 Jan. 6; 311 (5757):83-7.-   73. Manavalan J S, Kim-Schulze S, Scotto L, Naiyer A J, Vlad G,    Colombo P C, et al. Alloantigen specific CD8+CD28− FOXP3+T    suppressor cells induce IL T3+ILT4+ tolerogenic endothelial cells,    inhibiting alloreactivity. Int Immunol. 2004 August; 16(8): 1055-68.-   74. Jiang L, Barclay A N. New assay to detect low-affinity    interactions and characterization of leukocyte receptors for    collagen including leukocyte-associated Ig-like receptor-1 (LAIR-I).    Eur J Immunol. 2009 April; 39(4):1167-75.-   75. Suciu-Foca N, Feirt N, Zhang Q Y, Vlad G, Liu Z, Lin H, et al.    Soluble Ig-like transcript 3 inhibits tumor allograft rejection in    humanized SCID mice and T cell responses in cancer patients. J    Immunol. 2007 Jun. 1; 178(11):7432-41.-   76. Vlad G, D'Agati V D, Zhang Q Y, Liu Z, HoEK, Mohanakumar T, et    al. Immunoglobulinlike transcript 3-Fc suppresses T-cell responses    to allogeneic human islet transplants in huNOD/SCID mice. Diabetes.    2008 July; 57(7):1878-86.-   77. Cheng H, Mohammed F, Nam G, Chen Y, Qi J, Garner L I, et al.    Crystal structure of leukocyte Ig-like receptor LILRB4    (ILT3/LIR-5/CD85k): a myeloid inhibitory receptor involved in immune    tolerance. J Bioi Chern. 2011 May 20; 286(20): 18013-25.-   78. Cao W, Bover L, Cho M, Wen X, Hanabuchi S, Bao M, et al.    Regulation of TLR7/9 responses in plasmacytoid dendritic cells by    BST2 and IL T7 receptor interaction. J Exp Med. 2009 Jul. 6;    206(7):1603-14

1. An isolated anti-ILT3Fc monoclonal antibody A, B, C or D or anILT3-binding portion thereof.
 2. The monoclonal antibody of claim 1linked to a detectable label or to a cytotoxic agent.
 3. The monoclonalantibody of claim 2, wherein the detectable label is a radioisotope or afluorophore.
 4. The monoclonal antibody of claim 2, wherein thecytotoxic agent is a member selected from the group consisting ofTaxol®, Pseudomonas exotoxic fragment, cytocalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etopside, tenopside, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy antracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosteron,glycocorticoids, procain, tetracaine, lidokaine, propranolol, andpuromycin.
 5. The monoclonal antibody of claim 1, wherein the ILT3expressed on the surface of a cell comprises the extracellular domain ofILT3.
 6. A pharmaceutical composition comprising the isolated monoclonalantibody according to claim 1 or an antigen-binding portion thereof. 7.A method, comprising: (a) obtaining a biological sample from a subjectthat has or may have cancer that expresses ILT3 ligand on the cancercell surface, and a control sample from a normal subject; (b) contactingthe subject and control samples with a probe that selectively binds toILT3 ligand under conditions that permit the probe to bind to the ILT3ligand on the cancer cell surface; (c) determining the number orpercentage of cancer cells that are bound to the probe in the subjectand control samples, and (d) determining that the subject has the ILT3ligand-expressing cancer, if the number or percentage of cells bound tothe probe in the subject sample is significantly higher than thecorresponding number or percentage bound in the control sample.
 8. Themethod of claim 7, wherein the probe is a member selected from the groupconsisting of ILT3, ILT3Fc, extracellular domain of ILT3 or an ILT3ligand-binding fragment of the member.
 9. The method of claim 8, whereinthe probe is detectably labeled with a radioactive isotope or afluorescent label.
 10. (canceled)
 11. The method of claim 8, wherein theprobe is detected by binding to an anti-ILT3 antibody that is detectablylabeled.
 12. A method, comprising: (a) obtaining a biological samplefrom a subject that has or may have cancer that expresses ILT3 on thecancer cell surface, and a control sample from a normal subject; (b)contacting the subject and control samples with an antibody probecomprising an anti-ILT3 antibody, an anti-ILT3Fc antibody or a portionthereof that selectively binds to ILT3, under conditions that permit theantibody probe to bind to the ILT3 expressed on the cancer cell surface;(c) determining the number or percentage of cancer cells that are boundto the antibody probe in the subject and control samples, and (d)determining that the subject has the ILT3-expressing cancer, if thenumber or percentage of cells bound to the antibody probe in the subjectsample is significantly higher than the corresponding number orpercentage of cells bound to the antibody probe in the control sample.13. The method of claim 12, wherein the antibody is a polyclonal ormonoclonal anti-ILT3 antibody, anti-ILT3Fc antibody or an ILT3-bindingfragment of thereof.
 14. The method of claim 12, wherein the antibodyprobe is detectably labeled with a radioactive isotope or a fluorescentlabel.
 15. The method of claim 12, wherein the ILT3-expressing cancer ischronic lymphocytic leukemia (CLL) or AML with monocyticdifferentiation. 16-20. (canceled)
 21. A method comprising, a)identifying a subject in need of treatment for a cancer that expressesILT3 on its surface, and b) administering therapeutically effectiveamounts of an anti-ILT3 antibody, an anti-ILT3Fc antibody or a portionthereof that selectively binds to ILT3 or a combination thereof thatselectively binds to the ILT3 expressed on the surface of the cancercells, which antibody is linked either to an isotope that emitsradiation at a level that kills the cancer cell, or to a cytotoxicagent, thereby treating the subject for the cancer.
 22. The method ofclaim 21, wherein the anti-ILT3Fc antibody is a monoclonal antibody ofclaim
 1. 23. The method according to claim 21, wherein the cytotoxicagent is a member selected from the group consisting of Taxol®,Pseudomonas exotoxic fragment, cytocalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etopside, tenopside, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy antracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosteron,glycocorticoids, procain, tetracaine, lidokaine, propranolol, andpuromycin.
 24. The method of claim 21, wherein the cytotoxic agent isthe CD3 chain of the T cell receptor complex and cancer cell lysis isinduced by T-cell-mediated cytotoxicity against the cancer cell.
 25. Themethod of claim 21, wherein the cancer is AML with monocyticdifferentiation.
 26. A diagnostic kit for the detection of cancer cellsthat express ILT3 on their surface, comprising an isolated monoclonalantibody or an ILT3-binding portion thereof according to claim 1 orother monoclonal or polyclonal anti-ILT3 antibody, an anti-ILT3Fcantibody or a portion thereof that selectively binds to ILT3 orcombinations of antibodies.
 27. The kit of claim 26, wherein theantibody or portion thereof is detectably labeled either with aradioisotope or a label that can be visualized.
 28. (canceled) 29.(canceled)